Profilin regulates the stability and distribution of filamentous actin structures in all eukaryotic cells. This regulation is due, in part, to the ability of profilin to bind monomeric actin and catalyze nucleotide exchange, which can lead to an increase in filamentous actin. This activity is modulated by binding to phosphoinositides such as PIP2, which disrupts the profilin-actin complex. The binding of profilin to PIP2 also inhibits the phospholipase Cg catalyzed hydrolysis of PIP2 to the second messengers PIP3 and diacylglycerol. Thus profilin may serve to link the organization of the actin cytoskeleton to the phosphoinositide signal transduction pathway. The identification of profilins with different lipid binding and nucleotide exchange activities allows for regulation of the actin cytoskeleton. The binding of profilin to PIP2 will likely involve the interaction of basic surface residues with the acidic phospholipid headgroups, and may be facilitated by a conformational rearrangement placing hydrophobic side chains in the lipid interior. Birch pollen profilin binds actin but does not catalyze nucleotide exchange, Suggesting that modest alterations of profilin can confer nucleotide exchange activity. Structural information from high resolution X-ray diffraction and solid state NMR will help to identify residues involved in PIP2 binding and nucleotide exchange activity. The contribution of these residues will be tested by site directed mutagenesis and biochemical characterization. The in viva importance of species-specific and isoform-specific lipid binding and nucleotide exchange activities will be tested by heterologous expression of biochemically defined profilins in yeast and mammalian cells. These studies are likely to result in a structural mechanism for profilin sequestration by phosphoinositides and an explanation for the isoform specific interactions with PIP 2. The details of these interactions are central to understanding the connection between signal transduction pathways and cytoskeletal reorganization, and will represent the first structural analysis of a complex containing a molecule that regulates phosphoinositide/second messenger metabolism. The expression studies will test the importance of specific profilin activities in vivo, and identify species-specific differences in cytoskeletal regulatory mechanisms.